这项工作是由来自基金会法国，研究所国家德拉桑特的Et德拉RECHERCHE MEDICALE，皮埃尔和玛丽·居里大学和程序"INVESTISSEMENTS德前途报"ANR-10 IAIHU-06的资助。

所有的程序均按照欧盟的指导方针进行（指令63分之2010/ EU），并通过动物实验达尔文伦理委员会的批准。在这里我们描述我们在我们的实验室常规使用的方法，但是大多数步骤可以适于以匹配每个人的具体需要。 1.手术准备注:所有的切口和压点应与局部麻醉（利多卡因或布比卡因）反复渗透。本程序是终端，如果需要的无菌制剂几个修改应来实现。 <ol><li>麻醉与两个局部分离的腹膜内（IP）注射戊巴比妥钠（40mg / kg）和氯胺酮（50毫克/千克）大鼠。 </li><li>让全身麻醉下的动物去反复验证手术的麻醉平面达到（从头到脚没有反应捏）。放置老鼠在FEedback加热毯，并插入直肠探头保持37℃左右的核心温度。 </li><li>放置导管在腹腔，以促进麻醉剂的随后注射，避免反复针穿刺30射孔器官。 <ol><li>夹在头发上的小（〜2 - 3毫米），位于胃的下部右侧或左侧象限内的区域之上的区域。 注意:脱毛霜也可使用。 </li><li>做一个2 - 用锋利的剪刀或手术刀表层3毫米的切口。使用钝性分离，直至腹腔观察去除脂肪和肌肉层。 </li><li>插入约1 - 2厘米空腔小导管，并用手术胶水闭合伤口。 （ 如 ，2法语-对应于内径0.043毫米）:注意的直径和导管的总长度应是最小的，以减少死体积。聚氨酯管是最适合。 </li><li>做一个循环，并缝合Ť他导管皮肤固定到位。 </li></ol></li><li>安装气管插管人工呼吸过程中控制通风。 <ol><li>作为前一步骤，制备感兴趣的区域（1 - 2厘米在气管右柄以上），除去头发和切开皮肤。 </li><li>钝剖析脂肪和肌肉的第几层，然后移动唾液腺放在一边，轻轻解剖肌肉的最后一层暴露气管。 </li><li>小心地取出了气管组织和使用小镊子下方滑动线程。做一个外科医生的结与线程，但不紧张呢。 </li><li>切开2软骨环之间的横向气管。拭子血液在气管如果有的话。 </li><li>插入带有适当的直径的气管导管，拧紧线程的结，以确保管稳定。为了更稳定的线程可以进一步在更高点气管管的连接。 </li><li> SuturE中的伤口闭合或使用手术缝合钉。这里避免手术胶水如果气管套管打算重复使用。 </li></ol></li><li>在立体框架安装动物和仔细监视以下生理变量:脑电图， 血氧饱和度，ETCO 2，心脏率（通过心电图，ECG）和内部温度。监控和​​调整这些变量来麻醉和生理状态的适当深度。具体地，补充麻醉用戊巴比妥钠的必要时小剂量（10毫克/千克）。 </li><li>上双眼应用眼膏，以避免干燥。剪辑过的头发头皮，做一个纵行切口（〜2厘米），切除结缔组织覆使用手术刀或刮匙头骨。 </li><li>请在感兴趣的地区的一个小（1.5〜毫米直径）与开颅牙钻。 注:在这里，初级躯体感觉皮层每桶领域的目标是（7 - 8 mm产品前到双耳线，4.5- 5.5毫米横向中线31）。反复冲洗散热。 </li><li>使用额外的细镊子轻轻地使硬膜一个小洞。颅环锯术来放置脑电图电极内保留一个为0.5 mM区域（见下一步）。永久保留皮层湿润用0.9％NaCl溶液（或人造脑脊髓液）。 </li><li>放置一个低阻抗（〜60千欧）银电极（脑电图电极）上硬脑膜，避免未包括的脑膜皮层区域，并将参考电极上的头部的另一侧的头皮肌肉。 </li><li>在这个阶段（最后戊巴比妥钠注射后30分钟），保持通过经由IP导管戊巴比妥钠（10至15毫克/千克/小时）或芬太尼（3-6微克/千克/小时）的重复注射麻醉。前者将导致缓慢振荡，睡眠状，脑电图图案而后者将导致不同步，苏醒状，​​皮层轮廓。 </li><li>调整人工ventilation系统使得呼吸频率和音量类似于鼠的自发呼吸（正常范围70 - 115呼吸/分钟32）。然后，连接呼吸机的气管插管，并验证正确的胸廓膨胀（两侧）。如果不是这样，调整在气管轴的通风管道的位置。如果需要的话，吸存在于与连接到注射器或真空泵的导管气管的分泌。 </li><li>如果所有的生理变量32-35和脑电图29,36模式反映麻醉的稳定外科平面，做加拉明三乙基的肌内注射在每条腿瘫痪了大鼠，40毫克/公斤的第一注射，然后20毫克/千克每2小时19,29,36,50。 </li></ol> 2.细胞内记录<ol><li>拉玻璃微（微电极锐）用〜0.2微米的尖端比如它的电阻范围betweeñ50和80MΩ一次充满2M乙酸钾（醋酸钾）。 </li><li>放置在一个特定的保持器的吸管用银/氯化银（银/氯化银）丝（通过头部阶段）的移液管溶液到细胞内放大器连接。保持器应附到显微操纵。放置在大鼠的颈部肌肉一个Ag / AgCl参考电极。 </li><li>慢慢插入在大​​脑中的吸移管下到感兴趣的区域并通过监视电压验证其电阻下降响应于电流的步骤。使用动态更新（或扎普）按钮放大器，如果需要清除吸管。 </li><li>用棉签或合成吸收三角形干开颅（注意不要触碰脑电图或细胞内的电极）与硅橡胶或4％琼脂糖覆盖它来降低脑运动之前。 </li><li>降低1吸移管 - 2微米的步骤，直到其电阻增大接近的小区时。然后使用放大器的嗡嗡声功能penetra忒成神经元。 </li></ol> 3.诱导等电国家<ol><li>在这个阶段执行相应的实验方案（ 例如 ，射击反应，细胞内注入电流，电流-电压关系，感官刺激的反应），同时监视神经元的膜电位的脑电图和生理变量。 </li><li>确保细胞内电极是稳定的细胞通过在整个记录会话分析两个膜电位和动作电位特性的稳定性。如果没有，不去下一个步骤，并再次等待，直到记录变得稳定或查询其他神经元。 </li><li>注入戊巴比妥钠的高，但红外致死量（〜90毫克/千克，可低至从戊巴比妥初始条件35毫克/公斤，并为从所述芬太尼初始条件155毫克/千克）经由IP线路。 注意:在15 - 20分钟细胞内和脑电图WAveforms应减速慢行间歇电气沉默以瞬时达到所谓的"突发抑制"配置文件37,38，其折叠逐步向一个完整的等电位状态。据预计，心脏率显著减慢（由约10 - 20％），但血氧饱和度和ETCO 2应该保持相对稳定。 </li><li>如果未达到等电状态，注入戊巴比妥钠的少量（〜步骤3.1剂量的10％）。如果仍不能达到国家电增加更多的麻醉剂之前，请等待15分钟。 </li><li>重复实验方案进行比较的网络动态特性所记录的神经元的综合性能的影响。 注意事项:注射麻醉剂后停止，电气大脑活动应充分3至4小时内恢复。 </li><li>在实验结束时，注入戊巴比妥钠致死剂量（200毫克/千克，IP）来安乐死大鼠。 </li></ol>

The authors have nothing to disclose.

我们在这里描述在两个网络和细胞水平在体内自发脑电活动抑制的新方法。这个过程导致了极端的大脑状态，被称为电昏迷41。从临床观点来看，这样的大脑电活动是最严重的异常，可以在脑电图中看到。它主要以不可逆转的昏迷相关，所有患者要么垂死或继续在持续植物人状态42，但可以通过抑制中枢神经系统药物（如硫喷妥钠）的中毒时造成至少部分逆转，一个偶然的低温42或窒息性心脏骤停43。在我们的实验范例，等电状态逐渐由高剂量的戊巴比妥钠的全身注射，其中第一快速诱导的脑电图频率含量的减少，那么，"突发抑制"模式实现41,42，最终导致一个完全平坦的脑电图。在细胞内水平，自发性活动的消失遵循具有去极化的同时减少与超极化膜电位波动相似的时间过程。因此，可以假设戊巴比妥钠注射第一增加突触抑制传输导致减少皮层神经元击发活性，兴奋性和抑制性突触传递终于导致等电脑电图和细胞内活动29,44的逐步取消。从主动到等电位脑电图模式类似的转换可以在其他麻醉剂的管理，如氯胺酮（个人观察）或异氟醚45,46下获得。 该过程可能会出现相对比较简单。然而，由于非常深昏迷诱导，维持内基本生理变量正常范围是在实验的成功至关重要。在ETCO 2的波动变化可以是粘液栓在气管形成的结果。在这种情况下，呼吸机应断开和粘液迅速吸出或通过气管套管一扫而光。此外，该制剂的机械稳定性为细胞内记录的关键。因此，特别应该努力通过主体相对于动物的仔细调节的头部，同时保持适当的气管导管对准，以减少血管和呼吸脉动，并通过施加琼脂糖或对开颅硅氧烷弹性体。它也有必要通过麻痹剂的注射，以避免自发肌肉收缩。最后，环境振动和电噪声应减少尽可能。其他出版物细节体内准备最佳允许稳定的细胞内或细胞膜片钳的必要步骤谁LE-细胞记录29,47-50。 脱钩神经元的内在膜性能和网络动态的能力是必不可少的解剖由单个神经元在他们的高度连接的环境下处理信息的机制。正如前言所说，以前专门的根本神经科学这一核心问题研究导致冲突的结果，部分是由于神经元和调查网络和各种实验条件的特定功能， 包括体外 VS 体内的准备，最终的使用不同的麻醉程序（见例如29,36,51）。我们建议，本方法可以用于验证和可能调和，从体内实验在体外制备和从还原获得的发现。事实上，它允许直接检查，并在相同的神经元，并在相同的实验程序的过程中比较传入神经突触输入不同的模式的影响，从唤醒状动力学完成不活动，在活的动物中的神经元综合性能。 此协议的一个显着特点是，一旦掌握了，它可以与其它的实验技术，如多站点表面和深度脑电图记录，基于调查基因编码荧光指示剂和大脑甚至血流动力学和代谢成像进行组合，来调查多维等电脑的性质。作为临床和诊断的观点，因为我们表明，神经元是仍然持久等电昏迷中激发的，这将是相关的，以测试皮质功能，例如感觉信息的处理中，沉浸在脑的这样一种病理状态的患者和动物模型不活动。

在没有任何环境刺激或行为的任务中，"静止"大脑产生可从头皮进行记录的电活动的连续流，如脑电图（EEG）波。此内源性脑活动的细胞内相关成分的特征在于，背景膜的电压波动（也称为"突触噪声"），这是由反映传入网络1,2的正在进行的活动兴奋性和抑制性突触电位的组合。这种自发活动的频率和振幅与警觉的不同状态而变化。阐明网络活动的单神经元的兴奋性和反应能力的影响是神经科学的3,4的主要挑战之一。 许多实验和计算研究探索持续突触活动对综合propertie功能的影响神经细胞的的。然而，由背景突触噪声影响不同的神经元的参数的作用仍然是难以捉摸的。例如，膜去极化的平均等级已经发现与感觉输入触发的动作电位的能力正5,6或负7-9相关。此外，虽然一些调查表明，膜电位的波动，从传入的突触输入的连续变化的数据流得到的，强烈地受到调制的其输入输出关系3,10-13增益影响单个神经元的响应，其他指示通过分流抑制介导的细胞膜输入电导变化是足以调节神经元增益无论膜的波动14,15的幅度。最后，在清醒的动物进行最近的研究强调如何在单个神经元感觉信息的处理需要依靠警惕一个的状态ND当前的行为需求16,17。 阐明一个给定的处理的在高度互连的系统中的功能性作用一个直接的策略是，以确定如何其缺乏特异性改变系统的运作。该方法已在神经科学研究利用实验病变或不同脑区18-21的灭活，或特定的离子通道22,23的药理学阻断广泛使用，例如。值得注意的是，已经在体内施加到推出功能连接和网络动态如何影响单细胞计算24-27。然而，迄今为止本地操作旨在阻断神经元的放电和/或扰乱它们的基本生物物理特性可以是部分有效的，被限制为相对小的脑体积28。 为了克服这些限制，我们开发了体内实验方法进行新的大鼠来比较记录在一个给定的大脑状态单个神经元， 即，嵌入在一个特定的网络的动态，到整个大脑突触活动29的完全抑制后所得的电生理特性。在控制条件下，可能产生两个不同的皮质动态。睡眠就像electrocorticographic（脑电图）模式是由中等剂量戊巴比妥钠注射引起。另外，小振幅的快波脑电图相媲美的大脑皮层的活动清醒状态（醒状图案）基本可以用芬太尼注射产生。接着，同时保持相同的脑电图和细胞内记录时，由高剂量的戊巴比妥钠，其特征在于等电脑电图和细胞内活性的全身注射得到的内源性脑电活性的完全沉默。因为这样一个极端的昏迷诱导可能产生致命的consequen生物功能CES上，生理变量的仔细和连续监测是必不可少的。因此，我们认真随后心脏跳动频率，潮气末CO 2浓度（ETCO 2）时，O 2饱和度（SPO 2）和大鼠的核心温度在整个实验。 我们评估期间使用锋利微电极，其特别适合于在体内长时间稳定记录这些不同状态的单神经元特性。此处描述的方法，可以与其它电和成像方法相结合，并且可以延伸到其他的动物模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>Centravet</td>
			<td>Pentobarbital</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 500</td>
			<td>Merial</td>
			<td>Imalg&#232;ne 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl&#160;</td>
			<td>Janssen-Cilag</td>
			<td>Fentanyl</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylocaine</td>
			<td>Centravet</td>
			<td>Xylovet</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallamine triethiodide</td>
			<td>Sigma</td>
			<td>G8134</td>
			<td></td>
		</tr>
		<tr>
			<td>ECoG amplifier</td>
			<td>A-M Systems</td>
			<td>AC amplifier, Model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular amplifier</td>
			<td>Molecular Devices</td>
			<td>Axoclamp 900A</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition interface</td>
			<td>Cambridge Electronic Design</td>
			<td>CED power 1401-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Data analysis software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 7</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Scientifica</td>
			<td>IVM-3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Puller</td>
			<td>Narishige</td>
			<td>PE-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC150F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire 0.125mm (intracellular recording)</td>
			<td>WPI</td>
			<td>AGT0525</td>
			<td></td>
		</tr>
		<tr>
			<td>Ag-AgCl reference</td>
			<td>Phymep</td>
			<td>E242</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire 0.25mm (ECoG recording)</td>
			<td>WPI</td>
			<td>AGT1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial respiration system</td>
			<td>Minerve</td>
			<td>Alpha Lab</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological parameters monitoring</td>
			<td>Digicare</td>
			<td>LifeWindow Lite</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Blanket</td>
			<td>Harvard Apparatus</td>
			<td>507215</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>15005-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5</td>
			<td>FST</td>
			<td>11295-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5SF</td>
			<td>FST</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>IP Polyurethane catheter - 0.43x0.69mm&#160;&#160;</td>
			<td>Instech</td>
			<td>BTPU-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon elastomere</td>
			<td>WPI</td>
			<td>KWIK-CAST</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td>NSK</td>
			<td>Y1001151 and P496</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical glue</td>
			<td>3M</td>
			<td>vetbond</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of an isoelectric brain state to investigate the impact of endogenous synaptic activity on neuronal excitability in vivo

顺便神经元过程信息既取决于它们的内在膜特性和对传入突触网络的动态。特别是，内源性产生的网络活动，这强烈变化作为警戒的状态的函数，显著调制神经元的计算。探讨自发性脑动态如何影响不同的单个神经元"综合性能，我们开发了包括由高剂量戊巴比妥钠的全身注射的方式在体内抑制所有大脑活动的老鼠了新的实验策略。皮质活动，通过组合电图（脑电图）和细胞内记录连续监测正在逐渐放慢，从而导致稳定的等电轮廓。这种极端的脑的状态，把老鼠进入深度昏迷，小心地通过在整个实验测量动物的生理常数监视。细胞内řecordings允许我们表征和比较嵌入到生理学相关皮质动力学，如在睡眠 - 觉醒周期遇到了同样的神经元的综合性能，并且当大脑完全沉默。

诱导和维持的等电脑状态是在体内实验过程的细腻。它已被证明是一种强大的工具，直接研究了神经元兴奋性和传递函数29的皮质的网络活动的影响。 图1示出了（ 图1A之前多参数监控，包括脑电图和重要的常量，动物的生理状态的）和之后（ 图1B）诱导的等电状态的。 <img alt="图1" src="/files/ftp_upload/53576/53576fig1.jpg" /> 图1.在控制条件等电生理参数监测。 A和B，在活跃的状态皮质（A）和潜艇脑电图（顶部痕迹）和生理参数的同时记录equent等电周期（B）中。核心温度（温度），ETCO 2和血氧饱和度在整个实验中基本稳定。心脏跳动速度，相比之下，递减后的等电状态的诱导（从382到349次/分），作为上看到心电图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53576/53576fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 时的控制会话，所述生理参数是类似于在健康和清醒动物32-35测定的等电状态的诱导后不受影响，除了心脏速率略微减慢（ 图1）。这 是因为缺氧39或高二氧化碳40可以显着地改变神经细胞的兴奋，因此可以在一个双头引入严重的偏差的重要点<html> Ÿ探索神经综合性能的大脑状态依赖调制。 我们从两个不同的初始条件模仿皮质动力学在睡眠的早期阶段（ 图2AA，左图）或醒来（ 图2AB，左图）中内源性生成的所得到的等电状态。这些活性状态要么被戊巴比妥钠（睡眠等）或芬太尼注射诱导（醒来一样）。在这两种情况下，高剂量的戊巴比妥钠的随后的注射导致了脑电图和自发性活动的同时记录的神经元（ 图2AA和b，右面板），因此称为等电的完全废除。抑制正在进行突触活动导致了神经元膜电位（ 图2B）的一个显著稳定超极化。 <html> T"FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/53576/53576fig2.jpg" /> 图2.膜电位的自发动力突触活动抑制的后果。 （A）睡眠状（A a）和苏醒样（A B）模式（左图），并在相应的后续电时代中脑电图（顶部痕迹）和细胞内活动（内部，底部的痕迹），同时代表录音（在抑制注射后指示的时间;等电，右图）。脑电图信号的时间 - 频率分析（对于0能量密度 - 50赫兹的频率范围内）是由色标表示。膜电位值（B）的概率密度（P）的 （VM，仓位大小0.5毫伏，10秒录音）从面板A.所示的神经元这个数字已被修改，经许可，摘自参考文献29。/ftp_upload/53576/53576fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 为了说明这一点极端大脑状态的功能的影响，我们提取等电神经元的被动和主动内在性能，并与相应的初始状态期间测量了比较。使用这种策略，我们已经表明，神经元可以发射动作电位响应于等电状态期间去极化电流，这表明在细胞内注射，他们甚至背景突触活性（ 图3AA和b，等电）的完全抑制之后仍然完全可激发。此外，我们发现神经元的传递函数，通过测量由去极化增加强度（FI关系）的电流的步骤引起的发射频率进行评估，为右移与初始活性的条件下，指示在神经元兴奋性弱的输入（ 图3B）的灵敏度的降低。相应的神经元的增益， 即，将FI曲线的斜率，保持不变或控制状态是睡眠-或唤醒型，分别为（ 图3B）时降低。令人惊奇的是，神经元的表观输入电阻没有显著在缺乏突触驱动的改性相比对照活性的条件（ 图3AA，b）中 。更多的成果，包括人口分析，并在积极和等电条件的神经元的时间放电模式的量化，是我们最初的论文29可用。 <img alt="图3" src="/files/ftp_upload/53576/53576fig3.jpg" /> 图3.对膜性能和投入产出关系的三个皮层活动模式的比较影响。 （A）Voltag<html>睡眠等时体感皮层神经元去极化和超极化电流脉冲（底部痕迹）电子反应（中间的痕迹）（A a）和苏醒样（A B）脑电图模式（最高记录）和突触活动的剥夺后（等电）。膜输入电阻（R M，数值表示）是由超极化电流脉冲注射（-0.4 NA）引起的电压降（灰色的痕迹，20个试验平均值）测量。 （B）的相应的FI曲线，提供在面板（A）中所示的神经元的传递函数。在射速是为了应对不断增加去极化强度的电流脉冲（200毫秒时间）测量。每个电流强度施加20次和相应的燃烧率的平均值。虚线表示（A）中所示的细胞反应。这个数字已被修改，经许可，摘自参考文献29。上传/ 53576 / 53576fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo at JoVE.com

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

在此过程中生理相关的脑状态和正在进行的电活动完全取消后，执行从单个神经元长期在体内的细胞内记录，导致大脑电状态。动物的生理常数过渡到人工昏迷状态期间仔细监测。

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响<em&gt;在体内</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

induction-an-isoelectric-brain-state-to-investigate-impact-endogenous

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

8.0K 次观看.  Inserm U1127. 在此过程中生理相关的脑状态和正在进行的电活动完全取消后，执行从单个神经元长期在体内的细胞内记录，导致大脑电状态。动物的生理常数过渡到人工昏迷状态期间仔细监测。 

视频: Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

创伤性脑损伤诱导受控皮质冲击的母模型

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...